Octopus‐derived antioxidant peptide protects against hydrogen peroxide‐induced oxidative stress in IEC‐6 cells

Abstract This study aims to find antioxidant peptides from octopus protein hydrolyzates and verify the protective effects against H2O2‐induced oxidative stress in IEC‐6 cells. After the alcalase hydrolysate was ultrafiltrated, purified by Sephadex G‐25 gel fractionation and semipreparative reversed‐phase high‐performance liquid chromatography (RP‐HPLC), 16 peptides were identified, and chemically synthesized. In particular, the peptides AQNY, AMMLAW, FEGAW, GGAW, VDTVVCVW, and VVCLW showed better oxygen radical absorbance capacity (ORAC) and ABTS radical scavenging capacity. Among them, the smallest‐molecular‐weight peptide GGAW exhibited the best antioxidant activity. Furthermore, GGAW protected IEC‐6 cells from H2O2‐induced oxidative damage by significantly reducing the generation of reactive oxygen species (ROS), malondialdehyde (MDA), and lactate dehydrogenase (LDH), and increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH‐PX), thereby improving cell viability. These results indicated that the peptide GGAW possessed the antioxidant capacity to prevent oxidative stress damage.

components in functional foods, cosmetics, and pharmaceuticals for health promotion.
Excessive production of reactive oxygen species (ROS) induced by oxidative stress damage is the main cause of intestinal diseases, such as gastroduodenal ulcers, inflammatory bowel disease, and colorectal cancer . Antioxidative peptides can scavenge ROS and free radicals by single-electron and hydrogen transfer, and chelation of prooxidant transition metals (Zou et al., 2016). Antioxidative peptides show cellular antioxidant activity, reduce oxidative stress biomarkers, increase the activity of multiple antioxidant enzymes, and regulate the levels of antioxidant molecules (Aguilar-Toala & Liceaga, 2020).
It was reported that peptides (NPYVPR, AVPYPQR, KVLPVPEK, and ARHPHPHLSFM) are identified in milk-protected Caco-2 cells against oxidative stress induced by H 2 O 2 by increasing the levels of both antioxidant molecules and antioxidant enzymes and reducing the production of ROS (Tonolo et al., 2018). In a similar study, Mirdamadi et al. found that three peptides (VLSTSFCPK, VLSTSFYPK, and STSFPPK) obtained from Kluyveromyces marxianus protein hydrolyzate could activate the Keap1-Nrf2 signaling pathway, which may lead to a decrease in lipid and protein oxidation and cell apoptosis and an increase in cell viability (Mirdamadi et al., 2021).
Octopus vulgaris is an economically important seafood species worldwide because of its rich nutrition, strong environmental adaptability, fast growth, and high conversion rate (Luo et al., 2021;Vaz-Pires et al., 2004). Octopus protein hydrolyzates obtained by treatment with various proteases were reported to exhibit antioxidant activities such as scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, preventing the bleaching of β-carotene, and protecting DNA against breakage induced by hydroxyl radical (Slama-Ben Salem et al., 2017). Tripeptide GEY was obtained from Octopus aegina mantle protein using gastrointestinal enzymes and possessed effective free radical scavenging in lipid peroxidation, DNA damage, and cellular destruction under stress conditions (Sudhakar & Nazeer, 2017). The alcalase hydrolyzate of Octopus ocellatus meat showed the highest scavenging effects against free radicals and hydrogen peroxide as well as the highest oxygen radical absorbance capacity and reduced the ROS production level in H 2 O 2 -treated hepatocytes, without cytotoxicity (Um et al., 2017).
However, the specific antioxidant peptide sequence from the octopus hydrolyzate and the antioxidant effects on oxidative stress remain to be evaluated.
In brief, the antioxidant activity of octopus hydrolyzates prepared with neutrase, alcalase, and papain was compared. Ultrafiltration, size-exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), and tandem mass spectrometry (MS/MS) were used for the separation, purification, and identification of the antioxidant peptides. Furthermore, the protective effects of the best antioxidant peptide on IEC-6 cells subjected to H 2 O 2induced oxidative damage were determined.

| Materials
Octopus was freshly obtained from the Huangsha aquatic products wholesale market (Guangzhou, China). Fresh octopus meat was separated, cut into small pieces, homogenized, and finally refrigerated (−20°C). Rat small intestinal epithelial cell-line (IEC-6) cells were purchased from American Type Culture Collection. Neutrase, alcalase, and papain were purchased from Pangbo Enzyme Co., Ltd.

| Preparation of octopus protein hydrolyzate
The octopus homogenate was mixed with twofold (g/ml) distilled water and then hydrolyzed separately using alcalase (55°C, pH 8.0), neutrase (50°C, pH 6.5), and papain (50°C, pH 6.5) at 3000 U/g for 5 h. The reaction was stopped by boiling the samples in a water bath at 100°C for 10 min. The hydrolyzates were centrifuged at 30,000 g (Avanti J-26S XP Centrifuge, BECKMAN COULTER, Inc.) for 30 min.
The supernatant was collected, concentrated, and freeze dried, resulting in the octopus protein hydrolyzate ON, OA, and OP from neutrase, alcalase, and papain hydrolysis, respectively. The protein content of hydrolyzate powder was determined according to the AOAC method. The degree of hydrolysis (DH) was measured by the o-phthaldialdehyde (OPA) method described by Nielsen et al. (2001).

| Determination of amino acid composition
The sample was hydrolyzed with 6 mol/L HCl in a nitrogen atmosphere at 110°C for 24 h. The amino acid composition was analyzed using a Hitachi 835-50 automatic amino acid analyzer (Hitachi Co.) after hydrolysis.
The Trp level was measured after hydrolysis with 4 mol/L LiOH in a nitrogen atmosphere at 110°C for 20 h. The obtained hydrolyzates were filtered through a piece of filter membrane with a 0.22 μm pore size and determined by HPLC.

| Analysis of antioxidant activity
The DPPH, ABTS assay, and ORAC values were conducted by the reported method (Agrawal et al., 2016;Wattanasiritham et al., 2016) with some modifications. Trolox solutions (100, 50, 25, 12.5, and 6.25 μmol/L) were used to establish the standard curve. The DPPH, ABTS activity, and ORAC values were shown as μmol TE/μg peptide using the standard curve established previously.
The ultrafiltration component with the highest antioxidant activity was suspended in distilled water (30 mg/ml) and purified by a Sephadex G-25 gel filtration. The gel column (300 × 45 mm) was eluted with distilled water using EZ Purifier III liquid chromatography (Shanghai Lisui Chemical Engineering Co., Ltd.) at a flow rate of 10 ml/ min and monitored at 220 nm. The major peak was collected and lyophilized for antioxidant assay analysis and RP-HPLC purification.
The fraction with strong antioxidant activity was then subjected to YMC-Pack ODS-A column (250 × 10 mm, I.D. S-5 μm, 12 nm) on an Agilent 1260 system (Agilent Technologies) for further separation.
The flow rate was maintained at 2.5 ml/min using eluent A (deionized water containing 0.1% trifluoroacetic acid, TFA) and eluent B (acetonitrile containing 0.1% TFA). The elution program was as follows: 1-10 min, 6% B; and 10-30 min, 6%-49% B. The isolated fractions were monitored at 220 nm. Eighteen fractions were collected, dried by nitrogen flow, and lyophilized for antioxidant activity assays.

| Identification of peptide sequences
The fractionated peptides with the highest antioxidant activity were Inc.). Only those peptides that presented "de novo" −10lgP above 20 and were similar to the protein sequences of octopus by BLAST were considered for further analysis.

| Peptide synthesis
The biological peptides were synthesized using the FMOC solidphase procedure by ChinaPeptides Co., Ltd. The purity of the synthetic peptides was verified to be higher than 95% by analytical HPLC equipped with an API150-ESI mass spectrometry system.

| Detection of ROS, MDA, LDH, SOD, and GSH-PX levels
IEC-6 cells were seeded at 2.5 × 10 5 cells/well after treatment as described in Section 2.8. Cells were harvested with trypsin and washed with PBS. Cellular ROS was detected by the probe fluoresceinlabeled dye H2DCFDA according to the method reported by Dai et al. (2020). The level of malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) contents were determined using the corresponding kits obtained from Nanjing Jiancheng Bioengineering Institute.

| Statistical analysis
All data were expressed as means ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) with the Bonferroni multiple-range tests using IBM SPSS 22.0 software. p < .05 was considered as significant.

| Antioxidant activity of octopus protolyzate
On the basis of the tricine SDS-PAGE results, the peptide pool composition of each hydrolyzate is reported in Figure 1a. The DH percentages of OA, ON, and OP after 5 h of incubation were 31.47 ± 0.78%, 30.70 ± 1.30%, and 26.87 ± 0.66%, respectively. Compared with the other hydrolyzates, OA, which had the highest DH and protein content, should be rich in low-MW peptides. The DH affects the length and amino acid composition of peptides, which can greatly influence antioxidant activity (Sila & Bougatef, 2016). As reported in Table 1, the ON and OA hydrolyzates showed a similar amino acid composition, whereas OP displayed a different amino acid composition.
Hydrophobic amino acids, Val, Ile, Phe, Lys, His, and Trp, frequently occurred in OA and ON, accounting for 26.21% and 26.20% of the total amino acids, respectively, whereas they were poorly expressed in OP (25.20%). In contrast, Glu, Pro, Gly, and Ala were the most abundant amino acid residues in OP. Moreover, the Fischer ratio of OA and ON was higher than that of OP, which was beneficial for higher radical scavenging activity (Lan et al., 2019).
The antioxidant activity results of DPPH, ABTS, and ORAC assays, as shown in Figure 1b, demonstrated that OA possessed the highest radical-scavenging abilities and potential antioxidant properties. These results are consistent with recent antioxidant studies that reported that the alkaline protease hydrolyzate from octopus protein exhibited the highest scavenging ability compared to octopus treated with other commercial enzymes (Um et al., 2017). Wang et al. (2021) reported that the DH was highly positively correlated with antioxidant activity. This finding also showed that the higher the DH value, the higher the antioxidant activity of OA, which contained more low-molecular-weight bioactive peptides. In addition, this result might be due to the more hydrophobic amino acids in OA.
It was reported that peptides with high antioxidant activity contain a high proportion of hydrophobic amino acids, which is considered the key factor in the ability of peptides to scavenge radicals (Zou et al., 2016). Moreover, a significant difference in the DPPH radical scavenging ability between ON and OA was observed (p < .05). The content of Phe and His in OA, ON, and OP accounted for 8.14%, 7.83%, and 7.64% (Table 1), respectively. Mendis et al. suggested that the peptide showed higher radical scavenging activity when it contained more Phe and His residues (Mendis et al., 2005).
This result was consistent with previous research stating that protein hydrolyzates with low molecular weights have higher electron transfer efficiency and can scavenge radicals more effectively (Chalamaiah et al., 2012;Wen et al., 2020). After ultrafiltration, lowmolecular-weight peptides were enriched, which exerted more effective antioxidant activity than the whole hydrolyzates.
F I G U R E 1 (a) Heatmap of the amino acid composition of each hydrolyzate and (b) antioxidant capacity of octopus protein hydrolyzate. Octopus protein hydrolyzate ON, OA, and OP was prepared by neutrase, alcalase, and papain hydrolysis, respectively. Different lowercase letters on the bar indicated significant differences (p < .05) between groups. The ultrafiltration fraction MW <3 kDa was further purified using Sephadex G-25, as shown in Figure 2b, and three fractions with decreasing molecular weight were collected. The DPPH, ABTS +· , and ROO · scavenging abilities of F3 were 1.05 ± 0.10 μmol TE/μg peptide, 1.89 ± 0.23 μmol TE/μg peptide, and 3.16 ± 0.24 μmol TE/μg peptide, respectively, which were significantly higher than those of F1 and F2 (p < .05; Figure 2c). The last fraction F3 with the smallest molecular weight peptides and free amino acids exhibited the highest DPPH, ABTS +· , and ROO · scavenging activity. These results were similar to the reports that separated antioxidant peptides using Sephadex G-25, and they both determined that the peptides in the last fractions presented the highest antioxidant activity (Jia et al., 2020;Zhang et al., 2018). Therefore, the F3 fraction was recommended as the major active component to be further investigated.
Next, RP-HPLC was used to further separate the F3 fraction

| Sequence and activities of the antioxidant peptides
The 16 peptides recognized from fractions 15#, 16#, and 17# are summarized in Table 2. The characterized peptides consisted of 4-9 amino acid residues and had molecular weights between 389.17 and 919.45 Da. All the peptides were chemically synthesized to evaluate their ABTS +· and ROO · scavenging activity. As shown in Table 2   0.00 ± 0.00 0.00 ± 0.00 a According to "PeptideRanker" (PeptideRanker (ucd.ie)).
due to the aromatic group stabilizing radicals through resonance or delocalization (Ghassem et al., 2017;Sila & Bougatef, 2016). Table 2, the peptide segments GGAW, AMMLAW, FEGAW, VDTVVCVW, and VVCLW with the presence of Trp at the C-terminus form the strongest conventional hydrogen bonds, which contribute to enhancing their antioxidant activity. In addition, previous studies demonstrated that Gly is a potential target site of free radicals due to the presence of only a hydrogen atom in its side chain (Wu et al., 2018;Yang et al., 2020). In particular, peptides containing Gly are more flexible in exposing the functional residues to free radicals, causing a significant increase in antioxidant activity (Wu et al., 2021;Zhong et al., 2021). The antioxidant activity of GGAW was strongly attributed to its sequence containing 50% hydrophobic amino acids, two Gly residues at the N-terminus, and Trp at the Cterminal position.

| Protective effects of GGAW against oxidative stress in IEC-6 cells
The H  Hydrogen peroxide can rapidly diffuse through cell membranes and trigger oxidative stress, producing large amounts of ROS, which lead to cell damage (Di Marzo et al., 2018). Excessive ROS attack the polyunsaturated fatty acids in biofilms to form the lipid peroxidation product MDA. The higher the MDA content, the more severe the cell oxidative damage (Bouzenna et al., 2017). When the cell membrane is damaged, LDH is released into the blood (Wiriyaphan et al., 2015).
As shown in Figure  was significantly inhibited in a dose-dependent manner (p < .05). In particular, after pretreatment with 200 μg/ml GGAW, MDA and LDH generations were significantly decreased, with a significant difference from the control group (p < .05). Similar results were found with the peptide IRW, and its protective effect against oxidative stress was attributed to Trp, which can inhibit intracellular ROS accumulation and block the ROS-activated mitochondria-mediated cell apoptosis pathway (Yi et al., 2017). These results indicated that GGAW effectively weakened the oxidative damage induced by inhibiting ROS accumulation.
In addition, intracellular antioxidases such as SOD and GSH-PX constitute the antioxidant defense system to eliminate excess ROS and combat and prevent oxidative stress in cells (Wang et al., 2016).
SOD catalyzes the dismutation of two superoxide molecules to form molecular oxygen and hydrogen peroxide which are further converted into harmless substances by other enzymes, such as catalase and peroxidases (Nordberg & Arner, 2001). As shown in Figure 4e, Hydrophobic amino acids Ala and Gly can provide H + and enhance synergy with other amino acids to display antioxidant properties . Previously studies have showed that some similar peptides, such as MKAVCFSL , IYVFVR (Zhang et al., 2019), and WVSPLAGRT (Bollati et al., 2022), contained more aromatic and hydrophobic amino acids and efficiently ameliorated the damage of oxidative stress.

| CON CLUS IONS
In this study, the octopus hydrolyzate prepared with alcalase showed higher antioxidant activity than the neutrase and papain hydrolyzates. After ultrafiltration and purification by Sephadex amino acids, antioxidant amino acid Trp at the C-terminus, and Gly-Gly at the N-terminus. Furthermore, GGAW prevented oxidative stress induced by H 2 O 2 by reducing the contents of ROS, MDA, and LDH while enhancing the activities of the intracellular antioxidant enzymes SOD and GSH-PX. These results indicate that GGAW has great potential to be developed into functional foods due to its antioxidant activity.

ACK N OWLED G M ENTS
The Engineering, Chinese Academy of Sciences (no. ISEE2021PY05).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study.

DATA AVA I L A B I L I T Y S TAT M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.